Cell signaling is universally controlled by reversible protein phosphorylation. In prokaryotes, phosphorylation on histidine and aspartate was long considered the main signaling mechanism, but phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) is now emerging as equally important. In Mycobacterium tuberculosis (Mtb), signaling through Ser/Thr phosphorylation is essential. Phosphorylation on Tyr, however, has not yet been described and is thought to be absent. We now for the first time show protein tyrosine phosphorylation in Mtb. We unambiguously detected 25 phosphorylation sites on 16 proteins in a single growth condition. We show tyrosine phosphorylation activity of two Mtb Ser/Thr kinases (STPKs), suggesting that some STPKs are dual specificity kinases. Further, we show that Tyr phosphorylation controls the overall activity of the essential STPK PknB. Because PknB is essential for Mtb growth, other STPKs broadly regulate Mtb adaptations, and tyrosine phosphorylation determines virulence in other bacteria, these findings have wide implications for Mtb pathogenesis. Here, we will identify the kinases and phosphatases that mediate Mtb protein phosphorylation on Tyr.